379
J . geia. Nicrobiol. (1964), 34, 379-388
Printed i n Great Britain
Composition of Cell Walls of Ageing Pseudomonas
aeruginosa and Salmonella bethesda
BY F. M. COLLINS
Department of Microbiology, University of Adelaide, Adelaide, Sou,th Australia
(Received 21 J u n e 1963)
SUMMARY
Anaerobic cultures of Pseudomonas aeruginosa died rapidly in the absence of nitrate and death was normally followed by extensive autolysis. A
mutant which did not undergo extensive autolysis was isolated. Anaerobic
cultures of Salmonella bethesda did not die or lyse even after prolonged
incubation. The protein and lipid content of the parent P . aeruginosa cell
walls altered during ageing in contrast to S . bethesda walls which did not
alter greatly as the organisms aged. The amino acid and amino sugar
content of the three strains was determined. The diaminopimelic acid,
glycine, alanine, glutamic acid, glucosamine, muramic acid and glucose
content of the parent cell walls decreased by 50 yo as the organisms aged.
The mutant strain of P. aeruginosa and S . bethesda walls showed no such
change in ageing. Chemical changes similar to ageing could be produced
in the cell walls of P . aeruginosa by incubation with an ‘autolysin’ obtained
from old cultures of the parent strain.
INTRODUCTION
Pseudomonas aerugkosa is a strict aerobe which can grow under anaerobic
conditions when nitrate is added to the medium to act as an alternative to oxygen
as hydrogen acceptor. Under these conditions rapid growth occurs until the exhaustion of the nitrate supply after which all growth ceases and the culture rapidly passes
into the decline phase. Such cultures afford an opportunity t o examine some of the
changes which occur when non-proliferating organisms are held in an otherwise
non-toxic environment.
Rapid death and extensive autolytic changes have previously been observed in
anaerobically incubated cultures of Bacillus subtilis (Nomura R: Hosoda, 1956).
Kaufmann & Bauer (1958) noted rapid lysis of anaerobically incubated cultures of
B . subtilis and isolated an autolytic enzyme from the medium. They suggested
that this ‘autolysin’ was released in an active form by the organisms when the
normal cellular respiration was inhibited. Lysis of several species of sporing bacilli
has been examined in some detail (Strange, 1959) and a bacterial lysozyme has
been shown to be involved in some instances (Richmond, 1959). The cell walls of
Gram-negative bacilli are more complex in nature than those of the Gram-positive
bacteria (Salton, 1958, 1960) and in general, lysozyme treatment of Gramnegative bacteria does not result in cell lysis except under special conditions
(Warren, Gray R: Bartell, 1955; Repaslte, 1958). A number of Gram-negative
bacteria undergo autolysis on ageing, but for the most part little information is
available on the mode of action of the enzymes involved. The similarities between
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F. M. COLLINS
the rapid death and autolysis observed in the anaerobic cultures of Pseudonzoims
aeruginosa and that reported for the corresponding anaerobically incubated Bacillus
cultures suggested that a similar mechanism may be responsible for both phenomena. An 'autolysin' has been found in the culture medium of old P.aerugzhosa
cultures and comparison of the composition of cell walls isolated from ageing
organisms with that of young cell walls treated with the enzyme showed similar
changes to occur in both.
METHODS
Organisms. Pseudomonas aerugiraosa N C m 6750 and Salvzoi&ella bethesda strain
Md. 2 (kindly supplied by Dr N. Atkinson, University of Adelaide) were grown on
nutrient agar slopes at 37" for 24 hr and stored at 4 O . Subcultures were prepared
from these slopes from time to time.
mutant strain of P . aeruginosa, which was
not subject to extensive autolysis on ageing, was isolated from an old broth culture
Of NCTC 6750.
Medium. The growth medium contained the following nutrients :acid hydrolysate
of casein (Oxoid Ltd., England), 5-0 g. ; sodium nitrate, 5.0 g. ; ammonium sulphate,
1.0 g.; magnesium sulphate, 0.1 g.; trace elements solution, 1.0 ml. (Meiklejohn,
1950); 0.067 M-2-amino-2-hydroxymethyl
propane-1,3-diol (Tris) buffer (pH ';.O>,
11. The medium was autoclaved a t 115" for 15 min.
CuZturaZ conditions. The medium was dispensed in 1 1. quantities in screw-capped
serum bottles. Each bottle was inoculated with approximately 10' viable bacteria.
The air was removed with a vacuum pump after the insertion of a sterile hypodermic needle through the rubber wad. A positive gas pressure was introduced by
returning sterile pure nitrogen into the bottle and removing the needle. The positive
pressure ensured that air did not leak into the cultures during the prolonged incubation period. All cultures were incubated a t 37".
Counting methods. Viable bacteria were counted by the method of Miles & Misra
(1938). Total counts were made by the Williams (1952) method.
Quantitative analyses. Total-nitrogen, protein (Folin-Ciocalteau method and
biuret method), total reducing sugar, total hexosamine and total phosphorus determinations were made by methods described by Kabat & Mayer (1961). Nitrate
was estimated as previously described (Collins, 1956).
Individual sugars were identified chromatographically after hydrolysis of the
walls with 2 N-sulphuric acid a t 100" for 2 hr. The descending chromatograms were
run in ethyl acetate + acetic acid + water (3+ 1+ 3 by vol.) for 18 hr a t 20". The
papers were sprayed with aniline phthalate reagent (Cramer, 1954) and the sugar
content of each spot estimated by the method of Baar (1954). Muramic acid was
estimated by the method described by Strange & Kent (1959).
Amino acids were identified, after hydrolysis of cell walls with 6 N-EICl at 105"
for 18 hr, by two-dimensional chromatography as described by Salton (1953). The
intensities of the colours obtained with ninhydrin were compared with those for
standard spots of known amino acids developed under identical conditions.
Lipids were estimated by the method described by Salton (1953).
Preparation of cell walls. Organisms were washed twice in saline and once in
distilled water and suspended in cold distilled water a t a concentration equivalent
to 20 mg. dry wt./ml. The cells were then exposed for 15 min. to sonic vibration in
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Composition of ageing cell walls
381
a Raytheon disintegrator with an output of 50 W. a t 9 kc./sec., the suspension being
kept a t 0" throughout the experiment. The cell walls were washed by the method
of Munoz, Ribi & Larsen (1959) and their purity checked by the examination of
palladium shadowed preparations with a Philips model E M 100 electron microscope.
The cell walls were suspended in distilled water and stored a t -20" until required.
Concentration of autolysinf r o m old cultures of Pseudomonas aeruginosa. Eight-day
anaerobic cultures of P . aeruginosa were freed from whole cells by centrifugation
at 8000 rev./min. for 30 min. The supernatant fluid was adjusted to pH 7.0 with
x-acetic acid. Solid ammonium sulphate was slowly added in the cold to a final
concentration of 3 ~The
. precipitate which formed was spun down, washed with
31x1-ammoniumsulphate and re-dissolved in 0-067 fiI-Tris buffer (pH 7.0). The preparation was dialysed against 0-067 &r-Trisbuffer (pH 7.0). The precipitate in the
dialysis sac was spun off and discarded. Nucleic acids were removed with protamine
sulphate (Korltes, del Campillo & Ochoa, 1950). The autolysin was re-precipitated
with 3 3i-ammoniuni sulphate and dialysed against 0.067 &i-Trisbuffer (pH 7.0).
After centrifugation the clear supernatant fluid was assayed for protein and diluted
with buffer until it contained 5 mg. protein/ml. Three ml. of the enzyme preparation
was added to the equivalent of 200 mg. dry wt. 24 hr P.aeruginosa NCTC 6750 cell
walls suspended in 5 ml. of 0.01 nr-Tris buffer (pH S.0) and the volume made up to
10 ml. with Tris buffer (pH 8.0). The preparation was incubated at Gofor 20 hr
and the cell walls were spun down a t 20,000 g for 30 min. The walls were washed
twice in cold 0.067 M-Tris buffer (pH 7.0) and stored in distilled water at - 20" until
required for assay.
RESULTS
Th,e growth of Pseicdoznorzas aeruginosa and Salrnoiiella bethesda in anaerobic cultures
Both the parent and the mutant strain of Pseudomonas aeruginosa grew vigorously
in the anaerobic cultures. A maximum viable population of 3-5 x lo9 organisms/ml.
was maintained until exhaustion of the nitrate supply (shown by an arrow in
Fig. 1) whereupon a rapid and extensive decline in viable count was regularly
observed (Fig. 1). Following the decrease in viable count the P . aeruginosa NCTC
6750 culture showed 40-50y0 lysis on ageing, whereas the mutant showed very
little lysis (5-10y0; see Fig. 1). The maximum viable population of Salmonella
bethesda remained constant at 4-5 x 108 organisms/ml. throughout the experiment
and the culture did not undergo any observable autolytic changes during ageing.
The extensive lysis observed in the older cultures of P . aeruginosa NCTC 6750
suggested the presence in those cultures of an autolytic enzyme and so lysis experiments with young washed suspensions in the presence of cell-free culture filtrates
were attempted. Preliminary experiments showed that some lytic activity was
associated with the fluid of old cultures; to obtain significant results i t was necessary
to first concentrate the ' autolysin' by ammonium sulphate precipitation.
Incubation of young resting cell suspensions of Pseudoinonas aeruginosa NCTC 6750
with the crude autolysin concentrate resiilted in the lysis of 40-50 yoof the organisms
in 10-15 min. Total counts confirmed that after 60 min. only 20 yo of the original
organisms were still intact. Control suspensions which were incubated with heated
autolysin (100" for 10 min.) showed little or no lysis during this time (Fig. 2). The
addition of ethylenediaminetetraacetic acid (EDTA) to the test mixture greatly
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F. M. COLLINS
382
increased the rate of lysis of the parent Pseudomonad and the mutant so that
60-70 yolysis was observed within 2-4 min. Control suspensions (containing EDTA
only) decreased by 10 yo over the 10 min. period. Ammonium sulphate precipitation of cell-free culture filtrates of aged mutant broth cultures yielded preparations
with little or no lytic activity for parent or mutant Pseudomonads either in the
presence or absence of EDTA. Lysis presumably resulted from the hydrolysis of
the cell wall by the autolysin and attempts were, therefore, made to determine the
possible substrates of the enzyme by making chemical analyses of the cell walls of
the ageing bacteria.
P. aeruginosa NCTC 6750
100
50
P. aeruginosa (mutant)
S. bethesda Md 2
1oc
5c
0
50
100
150
Time (hr)
200
Fig. 1. Growth curves of Pseudomonas aeruginosa NCTC 6750, P. aeruginosa (mutant)
and Salmonella bethesda in casein hydrolysate medium incubated anaerobically at 37".
The arrow indicates the time the nitrate supply became exhausted. x - x
= viable
count; g-@ = total count. Nitrate was still present in the S. bethesda culture at
192 hr.
Compositioii of ageing bacterial cell ualls
The details of the balance sheets for washed cell walls of the three organisms are
set out in Tables 1 and 4.
Total nitrogen and protein. The total-N content of the walls of the three organisms
accounted for 8-11 yo of the dry weight and showed very little change with age.
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383
Composition of ageing cell walls
The cell walls of young Pseudomonas aeruginosa NCTC 6750 cultures contained
50-60 mg. protejn/100 mg. dry wt. wall and this rose to almost 70 mg. in old
organisms. By contrast, the cell walls of the Pseudomonas mutant together with
I
I
2
I
4
6
I
8
I
10
Time (hr )
Fig. 2. Lysis of resting suspensions of Pseudomonas aeruginosa NCTC 6750 and P. aeruginosa (mutant) by crude autolysin in the presence of 0.01 M-Tris (pH 8-0) and 100 p g .
ethylenedianiinetetraacetic acid (EDTA)/ml. 0-0, P. aeruginosa NCTC 6750 + autolysin
+EDTA ; 0-0,P. aeruginosa NCTC 6750 + autolysin ; +-+ , P. aeruginosa mutant +
autolysin + EDTA ; x - x P aeruginosa mutant + autolysin ; n-A, P. aeruginosa +
EDTA ; V-V) P. aeruginosa control.
)
Table 1. Compositioqi of cell walls oJ' ageing bacteria
-4
ge
Total-N
Pseudomonas
aeruginosa
24
48
96
192
24
96
120
192
48
96
192
Lipid
*
Free
Total
(mg.1100 mg. dry wt. cell walls)
(hr)
24
Total
reducing
Protein* sugar
Total-P
NCTC
I
8.4
54
8.7
50
(i1
64
6750
Enzyme treated?
P. aeruginosa
(mutant)
-
5.4
15
15
12
4
-
63
7.6
10
10.1
10.5
70
72
69
70
6.3
5.0
16
14
11
13
77
63
63
8.7
6.0
7.2
12
11
10
10.9
Salmonella bethesda
5.2
8.0
*
t
-
__
Average of Folin-Ciocalteau and biuret determinations.
24 hr P. aeruginosa NCTC 6750 cell walls incubated at 45"for 20 hr in 0.01 M-Tris buffer (pH 8-0)
with the enzyme preparation.
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F. M. COLLINS
384
those of Salmonella bethesda, contained 65-70 mg. protein/100 mg. dry wt. wall
irrespective of the age of the cells (Table 1).
Amino acids. The amino acid composition of the cell walls altered significantly
as the cells aged (Table 2). Although the over-all recovery of amino acids increased
with age, considerable decreases were observed in the amounts of diaminopimelic
acid (DAP), alanine, glutamic acid and glyciize present in the older cell walls of the
parent strain. The amino acid composition of the cell walls of young mutant
Pseudomonads closely resembled that of the parent but there was no corresponding
decrease in the DAP, alanine, glutamic acid or glycine content as the organisms
aged (Table 3). The amino acid composition of the Salmonella bethesda cell walls
differed quantitatively but not qualitatively from that of the Pseudomonas aeruginosa
preparations.
Table 2. Amino acid cornposition of Pseudonionas aeru,ginosa NC'TC!6750 cell walls
Age of cell walls (hr)
A
I
24
Erizyme
treated*
1
88
40
192
24
(mg./100 mg. dry wt. cell walls)
Amino acid
Phenylalanine
Leucine and isoleucine
Valine
Tyrosine
Arginine
Lysine
Alanine
G1y cine
Serine
Glutamic acid
Aspartic acid
Diaminopiinelic acid
A
r-
7.8
2.9
6.0
2.2
3.0
3.3
0.9
1.1
2.5
2.6
0.6
0-8
7.5
7.2
11.0
11.4
5-0
-
6.6
14.4
2.8
5.7
11.6
3-0
*
7
9.0
3.5
2.6
3.4
0.6
0.9
4.2
6-3
4.0
4.7
9.6
1.7
9.5
3.5
6.3
2.4
1.7
2.4
0.9
0.8
3.1
2.0
2.8
0.9
1.1
3.3
6.6
3.4
3.2
11.4
1-2
5.6
4.1
4.6
10.9
0.9
As in Table 1.
Table 3. Amino acid coinposition of Pseudomonas aeruginosa ( tnu,taitt strain)
nnd Salmonella bethesda cell walls
P . aeruginosa (mutant)
I
A
S . bethesda
f
\
Age of cell walls (hr)
h
f
24
96
192
48
A
-'
-__
88
7
192
(mg./100 mg. dry wt. cell walls)
Amino acid
Phenylalanine
Leucine and isoleucine
Valine
Tyrosine
Arginine
Lysine
Alanine
G1ycine
Serine
Glutamic acid
Aspartic acid
Diaminopimelic acid
h
I
7.3
2.8
3.9
4.2
1.3
1.7
5.1
7.1
5.4
5.8
9.3
3.2
11.3
3.2
6.8
6.4
1.9
2.5
6.2
8.4
84
8.6
8.9
5.5
10.8
4.0
4.7
5.4
1.7
2.3
7.3
8.7
8.9
6.0
7-0
5.1
7
4.1
1 *6
2-0
2-5
0.4
0.5
4.3
5.5
2.6
4.9
14-5
2.1
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6.6
2.5
2-3
3-0
0.3
0-5
3.5
8.0
2.0
4.9
15.0
1.9
7.2
2.7
2.6
4.2
0.7
0.6
3.5
14.0
3.0
7.8
22.5
2.9
385
Composition of ageing cell walls
Carbohydrate. The details of the total reducing sugar content of the three cell wall
preparations are given in Table 4. Pseudomoizas aeruginosa NCTC 6750 showed a
decrease in total reducing sugar content with ageing whilst the cell walls of the
mutant did not show any ageing effect. However, the cell walls of the mutant
contained more reducing sugar residues than did the walls of the parent strain.
The Salmonella bethesda cell walls contained 18.5 mg. reducing sugar/100 mg. wall
and this decreased slightly with ageing (Table 4). The walls of all three organisnis
contained 1.5-2-0mg. of amino sugar/100 mg. wall, of which about half was muramic
acid (Table 4). The amino sugar content of P . aerugirzosa NCTC 6750 cell walls
decreased by 50 yo as the organisms aged. Little change in the amino sugar content
of the other two organisms was noted. Chromatographic identification of the
individual cell wall sugars and amino sugars disclosed the presence of glucose,
mannose, rhamnose, glucosamine and muramic acid (Table 4). The cell walls of
P. aeruginosa NCTC 6750 contained approximately equimolar amounts of glucose
and rhamnose. The glucose content of the walls decreased by 60 yoas the organisms
aged, whereas the rhamnose and the mannose content changed very little (Table 4).
The glucose and mannose content of the cell walls of the mutant was almost double
that of the parent and this offers an explanation for the higher reducing sugar
content observed with the cell walls of the mutant. The sugar content of the
S . bethesda cell wall was quantitatively quite distinct from both Pseudomonads
and there was no detectable alteration in sugar content on ageing.
Table 4. Carbohydrate and amino sugar composition of cell walls of age,ing
bacteria
Glucosamine
Age
(hr)
24
24
72
192
48
88
192
f
Pseudomonas
aeruginosa
NCTC
I
Glucose
Mannose Rhamnose
(mg.1100 mg. dry wt. cell walls)
Strain
24
64
88
192
Muramic
acid
A
0.7
0.8
-
6750
0-6
Enzyme treated*
P. aeruginosa
(mutant)
Salmonella
bethesda
tr.
0.7
2.9
2.7
1-8
1.1
\
0-3
0.3
0.3
0.3
2.2
2.0
2.0
1.8
0.6
0.3
1.9
5.0
4.4
3.4
0.6
0.7
0.5
3.0
3.0
2.8
1.0
3.9
-
4.8
4-1
3.7
3.3
3.0
9.6
8.2
8.1
0.4
0.8
-
0.9
*
As in Table 1.
-f Estimated only as total hexosamine.
tr. = trace.
Lipid and total phosphorus. The phosphorus content of the cell walls of the three
strains varied from 1to 2 mg./100 mg. wall and there was no change on ageing. The
free lipid content varied between 5.2 and 8-7 mg./100 mg. wall and did not alter
with ageing. However, the total lipid content of the parent pseudomonad walls
decreased by almost 2 5 % (Table 1).The lipid content of the walls of the mutant
and of Salmonella bethesda showed very little change on ageing. The total recovery
G. Microb.
25
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XXXIV
F. M. COLLINS
of wall material varied from 80 to 100 yo.The high recoveries for the mutant walls
were mainly due to the unexplained 15-20 mg./100 mg. wall increase in the protein
content of these walls. The incomplete recoveries for Pseudomonas aeruginosa
NCTC 6750 and S . bethesda suggest that there may be other undetected minor
components in these walls.
Autolysis of washed Pseudornonas aeruginosa cell walls
Incubation of young Pseudomonas aeruginosa cell walls with the crude autolysin
obtained from old cultures resulted in a change in opacity of 15-20% in 18 hr.
Chemical assays carried out on the hydrolysed cell walls disclosed changes in
composition similar to those recorded above for aged cell-wall preparations. The
protein content increased somewhat but the total reducing sugar was not apparently
affected (Table 1). However, chromatographic examination of the cell-wall hydrolysates disclosed that the amino acids DAP, alanine, glycine and glutamic acid
decreased by 50 yo(Table 2). Similarly, the glucosamine and muramic acid content
of the walls decreased sharply (Table 4). The total lipid content of the walls also
decreased considerably (Table 1). The composition of a control cell wall suspension
treated with boiled autolysin resembled, within experimental error, the initial
24 hr old preparation recorded in Table 1.
DISCUSSION
The observed lysis of the anaerobically incubated Pseudomonas aeruginosa
6750 was due to the presence of an extracellular autolysin. Cultures of the
mutant strain and SaEmoneZla bethesda did not produce detectable amounts of
autolysin in ageing cultures, although the mutant was still susceptible to lysis.
Thus the mutation appeared to involve the loss of ability to produce an extracellular autolysin, rather than the formation of a lysis-resistant cell wall. The mutant
cells were still strictly aerobic and were subject to a rapid and extensive degree of
killing when incubated under anaerobic conditions. The absence of subsequent
lysis of these cells suggests that the lethal and lytic processes observed in cultures
of P. aeruginosa NCTC 6750 are distinct and not directly inter-related.
The gross chemical composition of the cell walls of the three strains of bacteria
underwent a number of changes as the cultures passed through their growth phases,
The most interesting alterations occurred in the amino acid and amino sugar content
of the ageing cell walls of the parent strain. The over-all amino acid content of the
walls increased slightly as the cells aged. This was to be expected from the observed
increases in total protein. The coincidence of the sharp decline in the DAP, alanine,
glycine and glutamic acid content of the walls of the parent strain with the onset of
autolysis suggested a causal relationship between the two phenomena. Significantly,
the cell walls of neither the mutant strain nor Salmonella bethesda showed any
comparable decrease in these amino acids. I n view of the known importance of
these amino acids in the cell wall mucopeptides of other Gram-negative bacteria
(Salton, 1958, 1960; Brown, 1958) it seems reasonable to presume that the autolysin
present in the cultures of the parent strain removed part of the rigid mucopeptide
layer from the Pseudomonas aeruginosa cell walls so that cell lysis resulted. The
removal of at least 5 0 % of the muramic acid and glucosamine from the aged
NCTC
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Composition of ageing cell walls
P. aeruginosa NCTC 6750 cell walls was taken as confirmation that the entire mucopeptide moiety was affected. However, the Pseudomonas autolysin was not a
simple bacterial lysozyme since the crude autolysin preparation was unable to lyse
Micrococcus lysodeikticus suspensions.
The removal of complex lipid from the cell walls of the parent strain during ageing
suggested the presence of an esterase in the autolysin. Sierra (1957) demonstrated
the presence of several esterases in Pseudomonas aeruginosa cells and noted a
correlation between their presence in the culture medium and the subsequent
induction of cellular lysis. I n the present study, both the whole organisms and the
autolysin concentrate were found to have lecithinase activity when tested against
serum lecithin. The loss of lipid from the ageing cell walls of the parent but not the
mutant organism suggested that lysis of the cell was initiated only after some
lipid complex in the wall had been removed. This was confirmed by the increased
autolysis of P. aeruginosa following treatment of the cells with acetone or Teepol.
Increased autolysis following treatment of cells with lipid solvents has previously
been observed by Warren et al. (1955) and others. Presumably the removal of a
lipid moiety exposed the mucopeptide layer which could then be attacked by other
enzymes present in the autolysin. Thus it seems likely that the P. aeruginoso
autolysin consists of a mixture of a t least two enzymes and is distinct from the
autolysins produced by the Gram-positive bacteria. Further purification of the
autolysins of this and other Gram-negative bacteria would, therefore, be useful and
should reveal whether or not the autolysin of P. aeruginosa is typical of the enzymes
produced by other Gram-negative micro-organisms.
The author wishes to thank Professor D. Rowley for his help and advice during
this study. I am indebted to Mr H. Konczalla (Physics Department, University of
Adelaide) for taking the electron micrographs and to Mrs A. McAskill for technical
assistance.
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